Question 1

In DNA replication theory, which hypothesis proposes that parental DNA is broken into fragments and each daughter molecule contains interspersed mixtures of old and new DNA throughout both strands?

Accepted Answer

Introduction / Context: Before the Meselson–Stahl experiment, multiple models were proposed for how DNA duplicates. This question distinguishes among conservative, semi-conservative, and dispersive replication models. Given Data / Assumptions: The hypothesis described says parental DNA is fragmented. Daughter DNA strands are mosaics: both strands contain alternating old and new segments. Concept / Approach: Match the described mosaic pattern to the formal model names. The dispersive model uniquely predicts mixed old/new DNA along both strands after each round. Step-by-Step Solution: 1) Conservative: parental duplex remains intact; a wholly new duplex is made. Not a mosaic.2) Semi-conservative: each daughter duplex has one old strand and one new strand; strands are not internally mixed.3) Dispersive: both strands in daughters consist of interspersed old and new DNA fragments, matching the description. Verification / Alternative check: Meselson–Stahl density data excluded pure conservative after one generation and later excluded dispersive due to observed strand patterns after additional generations and nicking/denaturation tests. Why Other Options Are Wrong: Conservative and semi-conservative do not predict internal mixing of old/new along the same strand; “evolutionary theory” is not a DNA replication model. Common Pitfalls: Confusing “semi-conservative” (one old + one new strand) with “dispersive” (mosaic within each strand). Final Answer: Dispersive theory

Question 2

At the E. coli oriC, the initial melting of about 40–45 base pairs requires which minimal set of factors?

Accepted Answer

Introduction / Context: Initiation at the E. coli origin (oriC) proceeds through ordered steps: origin recognition, DNA unwinding (melting), helicase loading, and replisome assembly. The question asks for the minimal factors needed for the initial 40–45 bp origin melting. Given Data / Assumptions: Organism: E. coli, origin oriC. Proteins: DnaA (initiator), DnaB (helicase), HU (DNA-bending protein). ATP is available for DnaA activity. Concept / Approach: DnaA-ATP binds DnaA boxes within oriC, oligomerizes, and induces local strand opening in the AT-rich DNA unwinding element (DUE). HU facilitates bending and melting. DnaB helicase acts after melting, being loaded by DnaC. Step-by-Step Solution: 1) DnaA in the ATP-bound state recognizes oriC and multimerizes.2) HU stabilizes wrapped/bent DNA, lowering the energy barrier for unwinding.3) This DnaA-ATP + HU complex opens ~40–45 bp at the DUE.4) Only after opening is DnaB loaded (by DnaC) to expand the replication forks. Verification / Alternative check: In vitro reconstitution shows origin opening by DnaA-ATP with architectural proteins like HU; helicase loading is a subsequent step, not a prerequisite for initial melting. Why Other Options Are Wrong: Options lacking DnaA miss the initiator. Options including DnaB or polymerase III add factors needed after melting, not for the minimal opening step. Common Pitfalls: Equating “melting” with full fork establishment. Helicase action extends unwinding but is not required for the initial 40–45 bp opening. Final Answer: DNA + ATP + HU + DnaA

Question 3

In DNA replication, each daughter duplex contains one parental (old) strand and one newly synthesized strand. This replication mode is called:

Accepted Answer

Introduction / Context: The fundamental model of DNA duplication explains how genetic information is preserved across generations. The question asks for the name of the mode in which each daughter molecule inherits one old strand paired with one new strand. Given Data / Assumptions: Double-stranded parental DNA serves as template. New synthesis follows base-pairing rules. After replication, each daughter has one old and one new strand. Concept / Approach: This description matches the semi-conservative model verified experimentally by Meselson and Stahl using isotopic density labeling of DNA in E. coli. Step-by-Step Solution: 1) Each parental strand templates a new complementary strand.2) Resulting daughter duplexes are hybrid: old + new.3) Therefore, the correct term is “semi-conservative”. Verification / Alternative check: Density gradient centrifugation after one and two generations produced hybrid and light bands consistent with semi-conservative replication rather than conservative or dispersive models. Why Other Options Are Wrong: “Mutational” is not a replication mode; “discontinuous” describes lagging-strand synthesis mechanics; “antiparallel” refers to strand polarity, not the inheritance pattern. Common Pitfalls: Confusing mechanical terms (discontinuous) with inheritance models (semi-conservative). Final Answer: Semi-conservative

Question 4

What is the primary role of primase during DNA replication?

Accepted Answer

Introduction / Context: DNA polymerases require a free 3'-OH to begin nucleotide addition. Primase solves the initiation problem at both leading and lagging strands. Given Data / Assumptions: Replication fork contains helicase, primase, and DNA polymerases. DNA polymerase cannot start de novo; it extends from an existing 3'-OH. Concept / Approach: Primase (an RNA polymerase) synthesizes short RNA primers that provide the necessary 3'-OH for DNA polymerase to extend. Step-by-Step Solution: 1) Primase lays down an RNA primer complementary to template DNA.2) DNA polymerase extends from the primer, copying the template.3) On the lagging strand, primase must act repeatedly to start Okazaki fragments. Verification / Alternative check: Biochemical reconstitutions show loss of synthesis initiation when primase is omitted; adding primase restores initiation capability. Why Other Options Are Wrong: Removal of primers is done by nucleases and replacement polymerases; helicase unwinds DNA, not primase; proofreading is a function of 3'→5' exonuclease activity in certain DNA polymerases. Common Pitfalls: Assuming DNA polymerase can start without a primer or confusing primase with helicase. Final Answer: Synthesize a short RNA primer to initiate DNA synthesis

Question 5

Eukaryotic linear chromosomes face the end-replication problem. Which mechanism primarily resolves this limitation at chromosome ends?

Accepted Answer

Introduction / Context: Lagging-strand synthesis near chromosome termini cannot fully replicate the 3' ends after primer removal. This is known as the end-replication problem. Given Data / Assumptions: Linear eukaryotic chromosomes have telomeres composed of short repeats. Conventional DNA polymerases need a primer and cannot extend 3' ends without it. Concept / Approach: Telomerase, a ribonucleoprotein reverse transcriptase, uses its internal RNA as template to extend the 3' overhang, allowing completion of complementary strand synthesis and maintenance of telomere length. Step-by-Step Solution: 1) Telomerase binds the 3' end and aligns its RNA template with telomeric DNA.2) It adds repeat units to the 3' end via reverse transcription.3) DNA polymerase then fills the complementary strand using a primer synthesized by primase. Verification / Alternative check: Cells lacking telomerase show progressive telomere shortening and replicative senescence; telomerase reactivation stabilizes telomeres. Why Other Options Are Wrong: Deliberate shortening does not solve the problem; the entire chromosome is not copied from RNA template; “none” is incorrect since telomerase is the established mechanism. Common Pitfalls: Confusing telomerase RNA as a general genome template rather than a short repeat template at ends. Final Answer: Using the enzyme telomerase

Question 6

Which organism listed below lacks a classical linker histone H1 (has no canonical H1, only a divergent or absent equivalent)?

Accepted Answer

Introduction / Context: Histone H1 is a linker histone that binds at the DNA entry/exit points of the nucleosome and helps higher-order chromatin folding. Some organisms do not carry a canonical H1 ortholog. Given Data / Assumptions: Classical H1 is abundant in many metazoans and plants. Yeast chromatin is compacted primarily through core histones and other architectural factors. Concept / Approach: Identify species known to lack a canonical H1. Budding yeast Saccharomyces cerevisiae lacks a typical H1; it possesses Hho1p, a divergent protein that does not function as a standard linker histone across the genome. Step-by-Step Solution: 1) Consider model organisms: Xenopus and sea urchin nuclei are rich in H1 variants; Arabidopsis encodes several H1 isoforms.2) Budding yeast lacks a classical H1 and relies on alternative mechanisms for chromatin organization. Verification / Alternative check: Genomic and proteomic surveys show absence of a canonical H1 gene in S. cerevisiae and only a divergent Hho1 protein with limited linker-like roles. Why Other Options Are Wrong: Sea urchin, frog, and Arabidopsis have well-characterized H1 proteins and variants participating in chromatin compaction. Common Pitfalls: Assuming all eukaryotes share identical histone complements; fungi often deviate from metazoan norms. Final Answer: Saccharomyces cerevisiae

Question 7

During DNA replication, which enzyme primarily separates the two strands by breaking hydrogen bonds at the fork?

Accepted Answer

Introduction / Context: Unwinding parental DNA is essential to expose single-stranded templates for polymerases. The question targets the identity of the unwinding enzyme at replication forks. Given Data / Assumptions: Replication involves coordinated action of helicase, primase, and polymerases. The task is to break hydrogen bonds between complementary bases. Concept / Approach: Helicases are ATP-dependent motors that translocate along DNA and disrupt base pairing to create single-stranded regions. Step-by-Step Solution: 1) Helicase loads at the fork and uses ATP hydrolysis for movement.2) As it moves, it breaks hydrogen bonds and opens the duplex.3) Single-stranded binding proteins stabilize the exposed strands for replication. Verification / Alternative check: In vitro systems without helicase show minimal fork progression; adding helicase restores unwinding and synthesis. Why Other Options Are Wrong: DNA polymerase synthesizes DNA rather than unwinding; “strandase” is not a standard enzyme; “none” conflicts with well-established fork biochemistry. Common Pitfalls: Confusing helicase with topoisomerase; the latter relieves torsional stress but does not separate base pairs directly. Final Answer: Helicase

Question 8

Approximately what is the packing ratio (fold compaction) of DNA in a nucleosome fiber relative to naked DNA?

Accepted Answer

Introduction / Context: Chromatin organization starts with wrapping DNA around histone octamers to form nucleosomes, creating the “beads-on-a-string” fiber. The question asks for the approximate compaction achieved at this level. Given Data / Assumptions: Nucleosome core particle wraps about 147 bp of DNA around histones. Linker DNA connects adjacent nucleosomes to form a 10-nm fiber. Concept / Approach: Packing ratio is the linear compaction of DNA length after nucleosome formation compared to naked DNA. Empirically, the beads-on-a-string fiber yields roughly 6–7-fold compaction; the commonly cited value is about 7. Step-by-Step Solution: 1) Each nucleosome wraps ~1.7 turns of DNA.2) Considering linker segments, average compaction of the 10-nm fiber is near 7-fold.3) Therefore, the best approximate answer is 7. Verification / Alternative check: Biophysical measurements and electron microscopy of reconstituted chromatin consistently report ~7-fold compaction at the primary nucleosome level. Why Other Options Are Wrong: Values 6, 8, or 9 deviate from the commonly accepted approximation; 7 is the standard textbook number. Common Pitfalls: Confusing higher-order compaction (e.g., 30-nm fiber) with the primary nucleosome fiber; those yield greater packing ratios. Final Answer: 7

Question 9

In bidirectional DNA replication, how many replication forks are present in a single replication bubble?

Accepted Answer

Introduction / Context: Replication often initiates at an origin, creating a bubble from which synthesis proceeds in both directions. This tests knowledge of fork number in such a structure. Given Data / Assumptions: Replication is bidirectional in most bacteria and eukaryotes. A bubble forms as DNA unwinds locally around the origin. Concept / Approach: A replication fork is the Y-shaped junction where unwinding and synthesis occur. Bidirectional replication implies two forks moving away from the origin in opposite directions. Step-by-Step Solution: 1) Origin fires and DNA melts locally.2) Two forks assemble on either side of the origin.3) Forks move outward, enlarging the bubble until they meet neighboring forks. Verification / Alternative check: Autoradiography and modern sequencing-based assays detect symmetric progression from origins, consistent with two forks per bubble. Why Other Options Are Wrong: One fork would imply unidirectional replication; three or four forks do not describe a single bubble from one origin. Common Pitfalls: Confusing multiple nearby bubbles (from multiple origins) with the number of forks within one bubble. Final Answer: Two replication forks

Question 10

In DNA replication, short Okazaki fragments are synthesized on the lagging strand, leaving small nicks (unsealed phosphodiester gaps) between adjacent fragments. Which enzyme finally seals these nicks to create one continuous DNA strand?

Accepted Answer

Introduction / Context: During semi-conservative DNA replication, the lagging strand is copied discontinuously as multiple short DNA pieces called Okazaki fragments. After fragment synthesis and primer removal, the sugar–phosphate backbone still contains nicks. This question tests your understanding of which enzyme catalyzes the final joining step that restores backbone continuity. Given Data / Assumptions: Lagging-strand synthesis generates multiple Okazaki fragments. RNA primers are removed and gaps are filled with DNA. Residual nicks remain between adjacent fragments until a specific enzyme acts. Concept / Approach: The key reaction is formation of a phosphodiester bond between a 3'-OH and an adjacent 5'-phosphate at a nick. The canonical enzyme for this ligation in cells is DNA ligase, which uses an energy cofactor (ATP in eukaryotes and bacteriophages; NAD+ in many bacteria). Step-by-Step Solution: 1) Helicase unwinds the duplex; primase lays RNA primers.2) DNA polymerase extends from primers, generating Okazaki fragments.3) RNA primers are removed and replaced with DNA (e.g., by DNA polymerase I in bacteria).4) DNA ligase recognizes nicks and, via ligase-AMP intermediate, catalyzes phosphodiester bond formation to seal the backbone. Verification / Alternative check: Historically, discovery of Okazaki fragments was paired with demonstrations that ligase-deficient systems accumulate nicks, confirming ligase as the essential sealing enzyme. Why Other Options Are Wrong: DNA gyrase: relieves positive supercoils ahead of the fork; it does not seal nicks. RNA ligase: joins RNA; the nick here is in DNA. DNA polymerase I: fills gaps after primer removal but leaves a nick that must be sealed by ligase. Helicase (DnaB): unwinds DNA; no ligation function. Common Pitfalls: Confusing gap filling (polymerase) with nick sealing (ligase), or assuming topoisomerases repair breaks; topoisomerases manage supercoiling, not Okazaki nicks. Final Answer: DNA ligase

Question 11

In HIV-1, drug resistance emerges rapidly. Which specific property of its reverse transcriptase most directly explains the high mutation rate that fuels resistance to antiretroviral drugs?

Accepted Answer

Introduction / Context: HIV-1 evolves rapidly in patients, often selecting variants that resist antiretroviral drugs. Central to this adaptability is the fidelity of reverse transcriptase (RT), the enzyme that copies viral RNA into DNA. This item probes which molecular property of RT most directly accounts for the high mutation rate driving resistance. Given Data / Assumptions: HIV-1 RT synthesizes viral DNA from an RNA template. Mutations arise during genome replication. Drug resistance correlates with replication error rates. Concept / Approach: DNA polymerases with a 3'→5' exonuclease (proofreading) can remove misincorporated nucleotides, lowering error rates. HIV-1 RT lacks this proofreading function, so mismatches persist, producing a high mutation frequency that accelerates resistance selection under drug pressure. Step-by-Step Solution: 1) RT initiates DNA synthesis using a tRNA primer and viral RNA template.2) Without 3'→5' exonuclease proofreading, mispairs are rarely corrected during synthesis.3) The resulting proviral DNA incorporates mutations that can alter drug-binding sites.4) Under therapy, variants with reduced drug susceptibility expand, manifesting clinical resistance. Verification / Alternative check: Comparative biochemistry shows polymerases with proofreading (e.g., many replicative DNA polymerases) have far lower error rates than HIV-1 RT, aligning with observed rapid viral evolution. Why Other Options Are Wrong: AZT cost does not determine mutation rate; it is a socioeconomic factor, not an enzymatic property. RNase H degrades RNA in RNA–DNA hybrids; it does not set base-substitution fidelity. Low affinity for correct dNTPs would reduce processivity, not specifically increase substitution errors as observed. RT does not use ribonucleotides to synthesize DNA; it uses deoxyribonucleotides. Common Pitfalls: Confusing processivity or RNase H function with proofreading; assuming resistance arises mainly from recombination rather than frequent point mutations produced by error-prone synthesis. Final Answer: It lacks a 3'→5' proofreading exonuclease activity

Question 12

Chromatin higher-order structure: a ~30 nm fiber in which each helical turn contains about five to six nucleosomes is called what?

Accepted Answer

Introduction / Context: Eukaryotic DNA wraps around histone octamers to form nucleosomes (~10–11 nm fiber). These units can pack into higher-order fibers near ~30 nm diameter. One classic model with five to six nucleosomes per helical turn is the solenoid model. This question checks recognition of that terminology within chromatin organization. Given Data / Assumptions: Fiber diameter is approximately 30 nm. Each helical turn contains roughly five to six nucleosomes. Asks for the conventional name of this arrangement. Concept / Approach: Two principal 30 nm packing models are described: the solenoid (one-start helix with bent linker DNA) and the zigzag/two-start model (straighter linker DNA crossing the fiber). The stem specifies five to six nucleosomes per helical turn, aligning with the solenoid description. Step-by-Step Solution: 1) Identify fiber diameter: ~30 nm indicates higher-order nucleosome packing.2) Note given stoichiometry: five to six nucleosomes per turn.3) Map to canonical models: these details match the solenoid model.4) Conclude the correct term is “Solenoid”. Verification / Alternative check: Texts often contrast solenoid vs zigzag. The “five to six nucleosomes per turn” cue specifically signals the solenoid arrangement. Why Other Options Are Wrong: Halonoid: not a standard chromatin fiber term. Kinetoplast: a mitochondrial DNA network in kinetoplastids, unrelated to nucleosomal fibers. None of these: incorrect because a standard term exists. Zigzag two-start fiber: alternative model, but the turn/packing cue favors solenoid. Common Pitfalls: Assuming all 30 nm fibers are identical; in reality, multiple packing modes may exist and depend on ionic conditions and linker histone presence. Here, the numeric cue resolves the correct term. Final Answer: Solenoid

Question 13

Human genome composition: approximately what percentage of human nuclear DNA consists of repetitive sequences (including transposable-element–derived repeats and other repeats)?

Accepted Answer

Introduction / Context: The human genome contains large fractions of repetitive DNA. These include interspersed repeats from transposable elements (LINEs, SINEs such as Alu, LTR elements), segmental duplications, and satellite DNA. Understanding their approximate proportion helps contextualize genome size and annotation. Given Data / Assumptions: Question asks for a broad percentage range, not an exact single value. Repetitive content varies by definition and method, but standard textbooks report a substantial fraction. Concept / Approach: Classic estimates place repetitive content around half the human genome. Interspersed repeats alone account for roughly 45% by many annotations; adding other classes keeps the overall in the ~40–50% band used in foundational courses. Step-by-Step Solution: 1) Recall common textbook ranges for repetitive content in humans.2) Identify the offered ranges and select the one matching standard teaching.3) Choose 40 to 50% as the most widely accepted instructional range. Verification / Alternative check: Genome annotation resources frequently cite ~45% interspersed repeats; different pipelines can nudge the total but remain near this bracket. Why Other Options Are Wrong: 10–20% and 20–30% underestimate the known repeat content. 30–40% is closer, but still usually low relative to common figures. 60–70% overshoots typical accepted ranges for humans. Common Pitfalls: Confusing coding sequence fraction (~1–2%) with repetitive fraction; mixing nuclear vs mitochondrial DNA; assuming repeats are functionless—some have regulatory or structural roles. Final Answer: 40 to 50%

Question 14

Okazaki fragments are produced during lagging-strand DNA synthesis. On which strand do they occur, and which enzyme covalently joins the fragments into a continuous strand?

Accepted Answer

Introduction / Context: Replication forks copy the two antiparallel strands differently. The leading strand is synthesized continuously, whereas the lagging strand is synthesized discontinuously as Okazaki fragments that later must be covalently joined. This question checks that mechanistic pairing: which strand and which enzyme. Given Data / Assumptions: Okazaki fragments indicate discontinuous synthesis. Final joining requires ligation of the sugar–phosphate backbone. Concept / Approach: Okazaki fragments arise exclusively on the lagging strand because DNA polymerases synthesize 5'→3', yet fork movement forces discontinuous synthesis on one template. DNA ligase seals the nicks between fragments after primer removal and gap filling. Step-by-Step Solution: 1) Recognize that leading strand is continuous; lagging is discontinuous.2) Link “Okazaki fragments” with “lagging strand”.3) Identify the sealing enzyme as DNA ligase. Verification / Alternative check: Biochemical assays show ligase-deficient extracts accumulate unsealed fragments on the lagging template. Why Other Options Are Wrong: Leading strand, DNA polymerase: leading strand is not fragmentary; polymerase extends but does not seal nicks. mRNA, anticodons: irrelevant to DNA replication. tRNA, DNA polymerase: unrelated to fragment ligation. Lagging strand, DNA gyrase: gyrase relieves supercoiling, not ligation. Common Pitfalls: Confusing gap filling (polymerase) with backbone sealing (ligase); thinking both strands form Okazaki fragments—only lagging does. Final Answer: Lagging strand, DNA ligase

Question 15

During replication, helicase unwinding increases positive supercoiling ahead of the fork. Which protein primarily causes this overwinding, and which enzyme removes it to allow fork progression?

Accepted Answer

Introduction / Context: DNA replication imposes topological stress. As helicase opens the duplex, positive supercoils accumulate ahead of the fork, potentially stalling progression. Topoisomerases resolve this stress. This question asks you to pair the cause of overwinding with the enzyme that relieves it. Given Data / Assumptions: DnaB is the primary replicative helicase in many bacteria. Positive supercoils arise ahead of the fork during unwinding. DNA gyrase is a type II topoisomerase that can introduce negative supercoils and remove positive supercoils. Concept / Approach: Helicase action is the proximate cause of overwinding (rotational constraint ahead of the fork). Gyrase transiently breaks both strands, passes another segment through, and reseals, thereby relaxing positive supercoils and enabling fork movement. Step-by-Step Solution: 1) Identify the unwinding enzyme: DnaB helicase.2) Recognize topological consequence: positive supercoils ahead of the fork.3) Assign the resolving enzyme: DNA gyrase removes positive supercoils. Verification / Alternative check: Inhibition of gyrase (e.g., by quinolones) causes replication fork arrest due to unresolved supercoils, confirming its role. Why Other Options Are Wrong: DnaB, DNA polymerase: polymerase does not resolve supercoils. DnaA, DNA gyrase: DnaA initiates origin melting; it is not the main cause of ongoing overwinding at the fork. SSB, DnaA: SSB stabilizes single strands; neither resolves supercoils. Primase, DNA ligase: primer synthesis and nick sealing do not manage topology. Common Pitfalls: Confusing initiation (DnaA) with elongation dynamics; assuming ligase or polymerase relieve torsional strain—this is topoisomerase work. Final Answer: DnaB (helicase), DNA gyrase (topoisomerase II)

Question 16

Density-gradient centrifugation in cesium chloride: DNA labeled with heavy nitrogen (15N) will band where relative to DNA containing light nitrogen (14N)?

Accepted Answer

Introduction / Context: CsCl density-gradient centrifugation separates macromolecules by buoyant density. Classic Meselson–Stahl experiments used 15N labeling to distinguish heavy vs light DNA and demonstrate semi-conservative replication. This question asks where 15N-labeled DNA bands relative to 14N DNA. Given Data / Assumptions: 15N increases DNA buoyant density compared to 14N. In a stable CsCl gradient, denser species migrate to positions of higher density (farther from the axis of rotation). Concept / Approach: Heavier (higher density) molecules band closer to the bottom of the tube (i.e., farther out in a swinging-bucket rotor). Therefore, 15N-DNA bands below 14N-DNA. Step-by-Step Solution: 1) Recall that buoyant density determines equilibrium position.2) 15N substitution increases DNA density.3) Thus, the 15N band forms lower than the 14N band in the gradient. Verification / Alternative check: Published density values show a measurable shift (~0.016 g/ml) between 15N and 14N DNA, yielding resolvable bands. Why Other Options Are Wrong: Above: would imply lower density, which is false for 15N DNA. Co-incident: contradicts the density difference. “All of these”: rotor speed influences resolution, not which one is denser. Meniscus: banding occurs at the density-matching point, not at the surface. Common Pitfalls: Confusing tube orientation; in most diagrams, “below” denotes the higher-density region toward the outer radius. Final Answer: Below the 14N DNA band

Question 17

Which enzyme catalyzes the 5'→3' elongation of a new DNA strand during replication?

Accepted Answer

Introduction / Context: Multiple enzymes coordinate at the replication fork. Identifying which one directly adds deoxyribonucleotides to a growing DNA chain is foundational knowledge for molecular biology. Given Data / Assumptions: Question targets the catalytic polymerization step. Standard fork proteins act with distinct roles: unwinding, priming, elongating, ligating, and topology control. Concept / Approach: DNA polymerases catalyze nucleotidyl transfer: the 3'-OH of the primer strand attacks the alpha-phosphate of an incoming dNTP, releasing pyrophosphate and forming a phosphodiester bond, extending DNA 5'→3'. Step-by-Step Solution: 1) Determine which function is “elongation of DNA”.2) Match to enzyme class: DNA polymerase performs template-directed addition of dNTPs.3) Conclude DNA polymerase is the elongating enzyme. Verification / Alternative check: In vitro, purified DNA polymerases synthesize DNA on primed templates; other fork proteins cannot catalyze chain elongation alone. Why Other Options Are Wrong: Helicase unwinds duplex DNA. DNA ligase seals nicks after synthesis. Primase synthesizes short RNA primers. Topoisomerase I relaxes supercoils; no chain elongation. Common Pitfalls: Confusing primase (primer synthesis) with polymerase (DNA extension). Final Answer: DNA polymerase

Question 18

Sanger chain-termination DNA sequencing requires deoxyribonucleoside triphosphates for synthesis. Which of the following are needed as the standard dNTP substrates?

Accepted Answer

Introduction / Context: In Sanger sequencing, DNA polymerase extends a primer using normal deoxyribonucleotides (dNTPs) while occasional incorporation of a dideoxynucleotide (ddNTP) terminates extension. Understanding the role of both dNTPs and ddNTPs is essential. Given Data / Assumptions: The method is “chain-termination”. Standard dNTPs drive processive synthesis; ddNTPs cause termination when incorporated. Concept / Approach: Polymerization requires the full set of dNTPs: dATP, dTTP, dGTP, and dCTP. ddNTPs are added at limiting ratios to terminate chains at specific bases. Without dNTPs, polymerase cannot extend, and sequencing would fail. Step-by-Step Solution: 1) Identify the substrates for normal extension: all four dNTPs.2) Recognize ddNTPs are terminators, not the main substrates.3) Conclude that dATP, dTTP, dGTP, and dCTP are required. Verification / Alternative check: Standard protocols specify a mixture of all four dNTPs plus small amounts of labeled or unlabeled ddNTPs. Why Other Options Are Wrong: dATP only, or subsets like dGTP and dCTP, or dTTP only: incomplete; synthesis across diverse templates would stall at missing bases. Only ddNTPs: would halt immediately; no readable ladders form. Common Pitfalls: Confusing the role of ddNTPs (terminators) with dNTPs (building blocks); overlooking that balanced dNTP pools are critical for uniform read lengths. Final Answer: All of these (all four dNTPs are required)

Question 19

Reverse transcriptase is a viral polymerase used by retroviruses. What product does it synthesize from an RNA template?

Accepted Answer

Introduction / Context: Reverse transcriptase (RT) is central to the retroviral life cycle. It converts the single-stranded viral RNA genome into DNA that integrates into the host genome. Knowing the direction of information flow catalyzed by RT is a core concept. Given Data / Assumptions: Template is RNA. Enzyme is reverse transcriptase. Product is a DNA copy to form a provirus. Concept / Approach: RT performs RNA-dependent DNA polymerization, creating DNA complementary to the RNA genome. It also has RNase H activity to degrade RNA in RNA–DNA hybrids and, subsequently, DNA-dependent DNA polymerase activity to synthesize the second DNA strand. Step-by-Step Solution: 1) The viral RNA genome enters the host with RT.2) RT uses the RNA as a template to synthesize complementary DNA (cDNA).3) RNase H degrades the RNA template in the hybrid.4) RT synthesizes the second DNA strand, yielding double-stranded DNA for integration. Verification / Alternative check: Antiretroviral nucleoside analogs target RT's polymerase function, inhibiting synthesis of DNA from RNA. Why Other Options Are Wrong: DNA from peptides: no enzyme translates peptides to nucleic acids. RNA from DNA: this is performed by RNA polymerase (transcription), not RT. RNA from RNA: RNA-dependent RNA polymerases, not classical retroviral RT. Protein directly from RNA: translation is done by ribosomes, not RT. Common Pitfalls: Assuming RT only makes the first strand; in fact, RT orchestrates both cDNA synthesis and second-strand synthesis via its polymerase and RNase H domains. Final Answer: DNA from RNA

Question 20

In molecular biology, what would happen to the ends of eukaryotic chromosomes after each round of DNA replication if telomerase were absent? Explain the end-replication problem and its consequence for telomeres.

Accepted Answer

Introduction / Context: Telomeres are repetitive DNA sequences at the ends of linear eukaryotic chromosomes. They protect coding DNA from erosion during replication. Telomerase is a ribonucleoprotein enzyme that counteracts inevitable losses caused by conventional DNA polymerases. This question probes understanding of the end-replication problem and why cells need telomerase to maintain chromosome integrity. Given Data / Assumptions: Linear chromosomes with telomeric repeats at both ends. DNA polymerases require a primer and synthesize DNA 5'→3'. RNA primers used on the lagging strand are removed after synthesis. No telomerase activity is available to extend 3' ends. Concept / Approach: Because DNA polymerase cannot start de novo and because primers at the very end of the lagging strand are removed, the last Okazaki primer leaves a gap that cannot be filled. This is the end-replication problem. Telomerase normally extends the 3' overhang using its internal RNA template, enabling standard polymerases to complete the complementary strand. Without telomerase, telomeres shrink with each S phase. Step-by-Step Solution: 1) During replication, leading strand synthesis can, in principle, reach the terminus, but the lagging strand is made in short fragments requiring primers.2) The terminal RNA primer on the lagging strand is removed, leaving a short single-stranded gap at the 5' end that DNA polymerase cannot fill.3) Each cell cycle therefore removes a small number of bases from the chromosome end.4) Telomerase normally extends telomeres to offset this loss; in its absence, loss accumulates.5) Progressive shortening eventually triggers DNA damage responses and replicative senescence. Verification / Alternative check: Experimental systems lacking telomerase show gradual telomere attrition across generations. Reintroduction of telomerase restores length maintenance, confirming causality. Why Other Options Are Wrong: Chromosomes would remain the same length due to DNA ligase action: Ligase seals nicks; it cannot add missing nucleotides without a template extension. Chromosomes would gradually lengthen: No known conventional replication process lengthens ends without telomerase. Ends would become circular: Circularization is not a normal response in eukaryotes and would require rare recombination events. Only mitochondrial DNA length would change: Mitochondrial genomes are typically circular and not telomerase dependent. Common Pitfalls: Confusing ligase or polymerase activities with telomerase, or assuming leading strand synthesis prevents all losses. Another pitfall is thinking end-shortening affects coding genes immediately; telomeres buffer losses first. Final Answer: Chromosomes would become progressively shorter.

DNA Structure and Replication Questions